Stress-activated cellular signal transduction mechanisms play a significant part in the pathogenesis of several important clinical conditions. Including ischemic injury (as occurs in heart attack and stroke), arthritis, septic shock, and the side effects of radiation and chemotherapy. The stress-activated protein kinases (SAPKs, also called JNKs) and p38 are mitogenic-activated protein kinase (MAPK) subgroups that are responsible for the activation of the activator protein-1 (AP-1) transcription factor complex in response to environmental stress and the inflammatory cytokines TNF and IL-1. Together with the nuclear factor-kB (NF-kB) pathway, the SAPKs and p38s represent major mechanisms of stress- and inflammation-induced gene expression. As with all MAPK pathways, the SAPKs and p38s are themselves regulated as part of MAPK-kinase-kinase (MAP3K) -> MAPK-kinase (MEK) -> MAPK core signaling molecules. While many such potential core modules have been identified, molecular basis for the regulation of these pathways has remained unclear in spite of the fact that dissecting MAP3K regulation is pivotal to the understanding of the physiologic roles of these pathways. The next phase of this on going project will be to focus on the regulation of two stress-activated MAP3Ks:MEK-kinase (MEKK)-1 and apoptosis signal-regulating kinase-1 (ASK1), by elements coupled to the TNF receptor (TNFR) complex. The principal investigator's ongoing studies indicate that MEKK1 is regulated by germinal center kinase-1 (GCK), a putative effector for TNF receptor-associated factor-1 (TRAF2). The investigator's studies also support the contention that ASK1 is a potential effector for TRAf2. He will use in vitro and in vivo biochemical methods to characterize the mechanism by which TRAF2 regulates the observed in vivo association between GCK1 and MEKK1. He will use biochemical and cell biological methods to determine if MEKK1 is activated by oligomerization mediated by GCK1 and or TRAF2. Finally, he will use in vitro and in vivo biochemical methods to determine if ASK1 is regulated by dissociation of an inhibitor protein, thioredoxin, and aggregation mediated by TRAF2. These studies will clarify further mammalian stress signaling pathways and contribute to the identification of novel anti-inflammatory drug targets and therapeutic strategies.